Two-stage protective groups for the synthesis of biopolymers

ABSTRACT

The invention relates to a method for the synthesis of a nucleic acid by gradual breakdown from protected synthesis building blocks carrying two-stage protective groups. The two-stage protective groups are split by means of a first exposure step and a subsequent chemical treatment step

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/EP02/07389, filed Jul. 3, 2002, and designating the U.S., whichclaims priority of 60/314,306 filed Aug. 24, 2001.

The present invention relates to a process for the synthesis ofbiopolymers by stepwise assembly from protected synthetic buildingblocks which carry two-stage protective groups. The two-stage protectivegroups are activated by a first illumination step and eliminated by asubsequent chemical treatment step.

The technology of light-controlled synthesis of biopolymers usingphotolabile protective groups opens up the possibility of producingbiochips in situ by synthesis from monomeric and oligomeric buildingblocks. Biochips have gained a very considerable importance for researchand diagnosis since they permit rapid and highly parallel processing ofcomplex biological problems. However, chips of the highest quality arerequired for this, so that there is a great interest in novel and moreefficient synthetic methods.

Photolabile nucleoside derivatives are used in the light-controlledsynthesis of nucleic acid chips. In this connection, the assembly of thechain of nucleic acid fragments normally takes place usingphosphoramidite synthons. The building blocks each carry a temporaryphotoprotective group which can be removed by incident light. Theprinciple of the synthesis provides for a cyclic sequence ofcondensation and deprotection steps (by light). The efficiency withwhich such a light-controlled synthesis can take place is determinedessentially by the photolabile protective groups used, in particular bythe efficiency with which they can be removed in the irradiation step.The photoprotective groups used to date for light-controlled synthesisare normally the protective groups NVOC (S. P. A. Fodor et al., Science251 (1991), 767 ff.), MeNPOC (A. C. Pease et al., Proc. Natl. Acad. Sci.91 (1994), 5022 ff.), DMBOC (M. C. Pirrung, J. Chem. 60 (1995), 1116ff.) and NPPOC (A. Hassan et al., Tetrahedron 53 (1997), 4247 ff.).Further known photolabile protective groups in nucleoside and nucleotidechemistry are o-nitrobenzyl groups and their derivatives (cf., forexample, Pillai, Org. Photochem. 9 (1987), 225; Walker et al., J. Am.Chem. Soc. 110 (1988), 7170). A further photolabile protective groupwhich has been proposed is the 2-(o-nitrophenyl)ethyl group (Pfleidereret al., in: “Biophosphates and their Analogues—Synthesis, Structure,Metabolism and Activity”, ELSEVIER Science Publishers B. V. Amsterdam(1987), 133 ff.) and derivatives thereof (WO 97/44345 and WO 96/18634).

The photolabile protective groups currently used for light-controlledsynthesis of nucleic acids (e.g. NVOC, MeNPOC, NPPOC) are generallydistinguished by a comparatively low absorption coefficient at thewavelength of the incident light. Irradiation of photolabile nucleosidederivatives normally takes place with high pressure Hg lamps at awavelength of 365 nm. The result of the low absorption coefficient ofthe photolabile protective group used at this wavelength is that only avery small proportion of the incident light can be utilized forexcitation of the molecules. In addition, the photolabile protectivegroups used are mostly colorless derivatives. The result of this in turnis that it is not possible during the synthesis to detect by simplespectroscopic methods whether the photolabile protective group is stillpresent on the nucleoside derivative or has already been partly orcompletely abstracted by the input of light. The abstraction process canthus be followed only with difficulty or not at all.

Muller et al. (Helvetica Chim. Acta 84 (2001), 3735-3741) describe aphotolabile protective group consisting of an MeNPOC group to which afluorescent coumarin derivative is coupled via an amino linker. Thisphotolabile protective group is employed for synthesizingoligonucleotides on DNA microarrays. Elimination of the photolabileprotective group takes place in a single step by irradiation.

The present invention provides a novel protective group with which theactivation step is induced by light and the actual deprotection step atthe reaction site takes place by chemical means, e.g. acid catalysis(FIG. 1). This novel protective group, and molecules carrying thisprotective group, can be employed for the synthesis of biopolymers.

One aspect of the invention is thus a process for the synthesis ofbiopolymers by stepwise assembly from synthesis building blocks whichcarry protective groups, with use of at least one synthesis buildingblock which carries a two-stage protective group which is activated byan illumination step and is eliminated by a subsequent chemicaltreatment step. The chemical treatment step preferably comprises atreatment with base, a treatment with acid, an oxidation, a reductionor/and an enzymatic reaction. The chemical treatment step particularlypreferably comprises an acid treatment.

In a particularly preferred embodiment of the invention, a derivatizedtrityl group is used as two-stage protective group. Trityl groups arenotable for their excellent ease of elimination, in particular bytreatment with acid. The two-stage trityl protective groups of theinvention are, by contrast, not acid-labile but are converted into anacid-labile form only after activation and elimination of one or morephotolabile components. Particular preference is therefore given to theuse of a synthesis building block which has a two-stage protective groupand has the general formula I:

where R₁ and R₂ are each independently selected from hydrogen, OR₃,O(CH₂)_(n)COOR₃ and NHZ, R₃ comprises a C₁-C₈alkyl group, a C₂-C₈alkenylgroup, a C₂-C₈alkynyl group or/and a C₆-C₂₀aryl group which mayoptionally have one or more substituents, X is the synthesis buildingblock, Y is in each case independently a photoactivatable protectivegroup, Z is an amino-protective group, n is an integer from 0 to 4, andwhere R₁ or/and R₂ may optionally be replaced by Y.

The alkyl, alkenyl and alkynyl groups may be linear or cyclic,straight-chain or branched. Preferred meanings of R₁ and R₂ arehydrogen, O-methyl, OCOO-methyl or a protected amino group, e.g. anamino group which has been converted into an amide function with asuitable carboxylic acid.

The invention also encompasses compounds which carry a plurality ofphotoactivatable protective groups, in particular compounds of theformula I in which at least one of R₁ or R₂ is replaced by aphotoactivatable protective group Y.

It is possible by varying the radicals R₁ and R₂ and substituting one orboth radicals by photoactivatable protective groups to adapt thelability to acid to the desired requirements.

The invention also encompasses compounds which carry one or morefluorescent groups, e.g. compounds in which Y is a fluorescentphotoprotective group or/and R₃ and Z are fluorescent groups (R. Ramage,F. O. Wahl, Tetrahedron Lett., 34 (1993), 7133) or molecules in whichthe fluorescence has been introduced by substitution on the tritylframework (J. L. Fourrey et al., Tetrahedron Lett., 28 (1987), 5157).

These fluorescent groups can be employed for the quality control ofbiochips as long as the excitation and emission wavelengths do notinterfere with the light-induced activation. This can take place forexample in biochip supports as disclosed in WO 00/13018.

The photoactivatable group Y of the two-stage protective group may inprinciple be selected from any known photoprotective groups such as, forexample, nitroveratryloxycarbonyl (NVOC),α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC),3,5-dimethoxybenzoincarbonate (DMBOC),2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl andderivatives thereof, 2-(o-nitrophenyl)ethyl and derivatives thereof.

If a plurality of photoactivatable groups Y are present, they may beidentical or different.

The photoactivatable component Y of the two-stage protective group canbe eliminated by an illumination step. Elimination of the group Yincreases the lability of the remaining protective group, so that it canbe eliminated by a subsequent chemical treatment step, while a two-stageprotective group which still contains the photoactivatable component Yis substantially resistant to elimination under such conditions.

The process of the invention is employed for the synthesis ofbiopolymers, with the biopolymer to be synthesized being assembledstepwise from a plurality of synthesis building blocks. The process isparticularly preferably employed for the synthesis of nucleic acids,e.g. DNA or RNA. However, it should be noted that the process is alsosuitable for the synthesis of other biopolymers such as, for example,peptides, peptide-nucleic acids (PNAS) or saccharides. The synthesisbuilding block may be a monomeric building block, e.g. a nucleosidederivative, or else an oligomeric building block, e.g. a dimer ortrimer, i.e. for example a di- or trinucleoside derivative. Thesynthesis building block is particularly preferably a phosphoramiditebuilding block. It is, however, also possible to use other nucleotidesynthesis building blocks, e.g. phosphate or phosphonate buildingblocks. A further possibility is also to employ linker or spacerbuilding blocks, e.g. as phosphoramidites, as synthesis building blocks.Particularly preferred linkers or spacers as carriers of two-stageprotective groups are described in DE 100 41 539.3.

The synthesis building blocks of the invention carrying a two-stageprotective group generally have more strongly lipophilic properties thanthe synthesis building blocks used to date in the prior art. Thesolubility of the synthesis building blocks, especially of thephosphoramidite synthons, in organic solvents is increased through thislipophilicity. The more homogeneous reaction management made possiblethereby leads to a higher coupling efficiency compared with the purephotolabile phosphoramidite synthons. Elimination of the colored tritylcation of the photoprotective groups of the invention, which has aconsiderably higher absorption coefficient than the elimination productsof other photodeprotection processes, also opens up the possibility ofdirect online process monitoring. This leads to an improvement in thequality control of biochips.

The trityl group of the photoprotective groups of the inventionadditionally makes selective functionalization of the 5′-hydroxyfunction possible. This leads to an enormous reduction in costs, becauseseparation of the 3′-5′ isomers is dispensed with.

Particular preference is therefore given according to the presentinvention to phosphoramidite building blocks which carry the two-stageprotective group on the 5′-O atom of the sugar, in particular of theribose or of the deoxyribose.

The synthesis of the biopolymers can be carried out in a conventionalway, for example on a solid phase. It is particularly preferred for aplurality of biopolymers carrying a different sequence of synthesisbuilding blocks to be generated in situ in the form of an array on asingle support.

Yet a further aspect of the invention are compounds of the generalformula I

where R₁ and R₂ are each independently selected from hydrogen, OR₃,O(CH₂)_(n)COOR₃ and NHZ, R₃ contains a C₁-C₈ alkyl group or/and a C₆-C₂₀aryl group which may optionally have substituents, X is a synthesisbuilding block for the synthesis of biopolymers or a leaving group, Y isin each case independently a photoactivatable protective group, Z is anamino-protective group, n is an integer from 0 to 4, and where R₁ or/andR₂ may optionally be replaced by Y.

Substituents of alkyl, alkenyl, alkynyl and aryl groups are preferablyselected from halogens, e.g. F, Cl, Br or I, OH, SH, —O—, —S—, —S(O)—,—S(O)₂—, NO₂, CN and NHZ, where Z is an amino-protective group. Thesubstituents may be present one or more times on the relevant radical.Aryl groups may also comprise ring systems with heteroatoms such as, forexample, O, N or/and S. Alkyl, alkenyl and alkynyl groups may be presentin straight-chain, branched-chain or cyclic form and optionally besubstituted by a C₆-C₂₀ aryl group, where the aryl group in turn maycontain substituents as indicated above. Substituents of aryl groups areselected for example from —OH, halogen, —C₁-C₄-alkyl, —O—C₁-C₄-alkyl,where the alkyl groups may be substituted as indicated above.

If X is a leaving group, it is a group which can be eliminated when thecompound (I) reacts with another compound. X is preferably a leavinggroup which can be eliminated by reaction with a nucleophile, whereappropriate in the presence of an auxiliary base such as pyridine.Preferred examples of X are: Cl, Br, I, tosylate, mesylate,trifluorosulfonate etc.

Diagrammatic representation of the protective group concept of theinvention is shown in FIG. 1. The synthesis building block (A) carries atwo-stage protective group (B-C). In a first illumination step, thephotolabile portion (B) of the protective group is eliminated. A secondchemical treatment step, e.g. by addition of acid, eliminates thechemically labile component (C) of the protective group, so that thesynthesis building block (A) is present in active form.

FIG. 2 shows an exemplary substance from a preferred class of two-stageprotective groups of the invention. It is based on the acid-labiletrityl group but contains in the p position of one phenyl radical aphotolabile component (in this case the NPPOC group) which reduces orcompletely blocks the acid-sensitivity of the trityl group. Illuminationand elimination of the photolabile component converts the protectivegroup into an acid-labile form, and it can subsequently be eliminated inthe presence of acid to release the unprotected synthesis buildingblock.

FIG. 3 shows two variants for the synthesis of a two-stage protectivegroup of the invention.

FIG. 4 shows two further variants for the preparation of synthesisbuilding blocks of the invention with two-stage protective groups.

FIG. 5 shows a third variant of the synthesis of two-stage protectivegroups, with two of the phenyl groups being substituted on the tritylgroup by a photolabile group.

FIG. 6 shows specific embodiments of carbonate derivatives of thetwo-stage protective group and

FIG. 7 shows a process for the synthesis of the S1 protective group.Starting from ethyl 4-hydroxybenzoate andtert-butoxydiphenylchlorosilane in the presence of imidazole it ispossible to introduce the silyl group quantitatively. Thesilyl-derivatized ester is reacted with the Grignard reagent ofbromobenzene to give 4-(tert-butoxydiphenylsilyloxy)triphenylcarbinol(S1). In an analogous manner,4,4′-dimethoxy-4″-(tert-butoxydiphenylsilyloxy)tritylcarbinol (S3, nofig.) is obtained by using bromoanisole.

FIG. 8A shows the synthesis of the S2 protective group.

The methoxy silyloxy derivative can be obtained, starting fromsilyl-protected 4-hydroxybenzophenone which is synthesized in analogy toethyl hydroxybenzoate in the presence of imidazole, by reaction with theGrignard reagent of bromoanisole to give4-tert-butoxydiphenylsilyloxytriphenylcarbinol (S2).

FIG. 8B shows a scheme for synthesizing S4/5, R=H or OMe.

The disilyloxy tritylcarbinol derivatives (S4,5) are prepared byreaction of a protected 4,4′-dihydroxybenzophenone which reacts eitherwith bromobenzene (R=H) or with bromoanisole (R=OMe).

FIG. 9 shows the synthesis of the S6 protective group.

The trisilyloxy tritylcarbinol variant (S6) is prepared either byreacting 4′-silyloxybromobenzene with ethyl 4′-silylbenzoate or byreacting 4,4′-disilyloxybenzophenone and 4′-silyloxybromobenzene.

The tert-butoxydiphenylsilyl group can be eliminated from thesilyl-protected compounds by reaction with TBAF for one hour; stoppingthe reaction by adding pyridine/methanol/water and pyridinium-DOWEX. Thederivatives can usually be reacted directly with Nppoc-Cl.

In the subsequent halogenation of carbinols, diverse properties emerge,which are caused by the variation in the electron-attracting andelectron-donating substituents in the para position.

Replacement of the alcohol by a halide takes place by the mechanism of1st order nucleophilic substitution. The result of this is that allprocesses proceed through a trigonal-planar carbenium ion. The stabilityof the cation, its structure and the possibility of delocalization of πelectrons are crucial for this mechanism. It is additionally stabilizedby electron-donating substituents and destabilized byelectron-attracting substituents. In this case, the stability of thecation is directly associated with the acid resistance of the protectivegroup. A more stable cation means that the corresponding protectivegroups are more acid-labile.

A number of standard processes used for halogenating triphenylcarbinolderivatives are described in the literature. Experience withpyrene-substituted dimethoxytritylcarbinols shows that extremelyelectron-rich compounds can be chlorinated quantitatively with acetylchloride in cyclohexane within minutes. It emerges that good conversionsare achieved with refluxing SOCl₂ for selected protective groups withelectron-attracting para substituents.

FIG. 10 shows the preparation of protected thymidine derivatives.

Lower yields are obtained for compounds with a plurality of Nppocgroups. It was possible by using refluxing acetyl bromide or SOBr₂ toprepare the halogen derivatives in the presence of a plurality of Nppocsubstituents on the analytical scale and react them without furtherworkup with thymidine.

FIG. 11 shows a further process for the synthesis of nucleotides whichcarry a two-stage protective group.

FIG. 12 and FIG. 13 show processes for the preparation of nucleotideswhich carry a two-stage protective group starting from rosolic acid.

Tri-Nppoc-tritylthymidine is obtained in a one-pot reaction startingfrom rosolic acid. It is possible to dispense with the use of costlyprotective groups in this approach. The tripivaloyltrityl derivative wasused as model compound for this type of reaction.

The reaction proceeds in a polar aprotic solvent at 50° C. withinminutes via an intermediate which then reacts further with thecorresponding nucleosides to give the tandem-protected derivatives. Thepreparation takes place on the preparative scale.

EXAMPLES Example 1 Synthesis of Tandem Protective Groups 1.14-(tert-Butoxydiphenylsilyloxy)triphenylcarbinol (S1)

0.61 g (25.00 mmol, 2.1 eq) of magnesium was suspended in 20 ml of THFin a heat-dried flask under argon. 6.28 g (40.00 mmol, 3.4 eq) ofbromobenzene were slowly added dropwise from a dropping funnel withcontinuous boiling. After the addition was complete, the mixture washeated with an oil bath to 85° C. and stirred for 15 min. The resultingmagnesium bromide was allowed to cool to 70° C., and 5.00 g (11.89 mmol,1 eq) of ethyl 4-tert-butoxydiphenylsilyloxybenzoate dissolved in 20 mlof THF were added dropwise and heated at 85° C. for a further 1.5 h. Themixture was then allowed to cool and stopped with saturated ammoniumchloride solution. For the extraction, 500 ml of ethyl acetate wereadded and extracted 2× with ammonium chloride solution (sat.) and 1×with sodium chloride solution (sat.). The organic phase was dried withsodium sulfate and evaporated. The residue was purified by columnchromatography on silica gel 40-63 μm with the eluent ethylacetate/n-hexane+1% TEA (1:7).4-(tert-Butoxydiphenylsilyloxy)triphenylcarbinol is obtained as a whitepowdery solid.

Yield: 5.75 g≈91% of theory.

R_(f) (ethyl acetate/n-hexane 1:7+1% TEA): 0.19

1.2 4,4′-Dimethoxy-4″-(tert-butoxydiphenylsilyloxy)triphenylcarbinol(S3)

2.78 g (114.14 mmol, 2.4 eq) of magnesium were suspended in 80 ml of THFin a heat-dried flask under argon. 26.68 g (142.67 mmol, 3.0 eq) ofbromoanisole were slowly added dropwise from a dropping funnel withcontinuous boiling. After the addition was complete, the mixture washeated with an oil bath to 85° C. and stirred for 15 min. The resultingmagnesium bromide was allowed to cool to 70° C., and 20.00 g (47.56mmol, 1 eq) of ethyl 4-tert-butoxydiphenylsilyloxybenzoate dissolved in100 ml of THF were added dropwise and heated at 85° C. for a further 2h. The mixture was then allowed to cool and stopped with saturatedammonium chloride solution. For the extraction, 500 ml of ethyl acetatewere added and extracted 2× with ammonium chloride solution (sat.) and1× with sodium chloride solution (sat.). The organic phase was driedwith sodium sulfate and evaporated. The residue was purified by columnchromatography by the step gradient method on silica gel 40-63 μm withthe eluent ethyl acetate/n-hexane+1% TEA (1:5 and 1:3).4,4′-Di-methoxy-4″-(tert-butoxydiphenylsilyloxy)triphenylcarbinol isobtained as a white powdery solid.

Yield: 25.65 g≈97% of theory.

R_(f) (ethyl acetate/n-hexane 1:3+1% TEA): 0.59

1.3 4-Methoxy-4′-(tert-butoxydiphenylsilyloxy)triphenylcarbinol (S2)

350.64 g (26.51 mmol, 1.2 eq) of magnesium was suspended in 20 ml of THFin a heat-dried flask under argon. 6.20 g (33.14 mmol, 1.5 eq) ofbromoanisole were slowly added dropwise from a dropping funnel withcontinuous boiling. After the addition was complete, the mixture washeated with an oil bath to 85° C. and stirred for 15 min. The resultingmagnesium bromide was allowed to cool to 70° C., and 10.00 g (22.09mmol, 1 eq) of 4-tert-butoxydiphenylsilyloxybenzophenone dissolved in 50ml of THF were added dropwise and heated at 70° C. for a further 2 h.The mixture was then allowed to cool and stopped with saturated ammoniumchloride solution. For the extraction, 250 ml of ethyl acetate wereadded and extracted 2× with ammonium chloride solution (sat.) and 1×with sodium chloride solution (sat.). The organic phase was dried withsodium sulfate and evaporated. No workup was carried out.4-Methoxy-4′-(tert-butoxydiphenylsilyloxy)triphenylcarbinol is obtained.

Yield: quantitative reaction

R_(f) (ethyl acetate/n-hexane 1:3+1% TEA): 0.58

1.4 4,4′-di(tert-Butoxydiphenylsilyloxy)triphenylcarbinol (S4)

0.20 g (8.28 mmol, 1.2 eq) of magnesium was suspended in 20 ml of THF ina heat-dried flask under argon. 1.63 g (10.35 mmol, 1.5 eq) ofbromobenzene were slowly added dropwise from a dropping funnel withcontinuous boiling. After the addition was complete, the mixture washeated with an oil bath to 85° C. and stirred for 15 min. The resultingmagnesium bromide was allowed to cool to 70° C., and 5.00 g (6.90 mmol,1 eq) of 4,4′-di-tert-butoxydiphenylsilyloxybenzophenone dissolved in 50ml of THF were added dropwise and heated at 70° C. for a further 2 h.The mixture was then allowed to cool and stopped with saturated ammoniumchloride solution. For the extraction, 250 ml of ethyl acetate wereadded and extracted 2× with ammonium chloride solution (sat.) and 1×with sodium chloride solution (sat.). The organic phase was dried withsodium sulfate and evaporated. The residue was purified by columnchromatography on silica gel 40-63 μm with the eluent ethylacetate/n-hexane+1% TEA (1:7). 4,4′-di-(tert-Butoxydiphenylsilyloxy)triphenylcarbinol is obtained as a white powdery solid.

Yield: 3.00 g≈54% of theory.

R_(f) (ethyl acetate/n-hexane 1:7+1% TEA): 0.27

1.5 4,4′-di(tert-Butoxydiphenylsilyloxy)-4″-methoxytriphenylcarbinol(S5)

0.20 g (8.28 mmol, 1.2 eq) of magnesium was suspended in 20 ml of THF ina heat-dried flask under argon. 1.94 g (10.35 mmol, 1.5 eq) ofbromoanisole were slowly added dropwise from a dropping funnel withcontinuous boiling. After the addition was complete, the mixture washeated with an oil bath to 85° C. and stirred for 15 min. The resultingmagnesium bromide was allowed to cool to 70° C., and 5.00 g (6.90 mmol,1 eq) of 4,4′-di-tert-butoxydiphenylsilyloxybenzophenone dissolved in 50ml of THF were added dropwise and heated at 70° C. for a further 2 h.The mixture was then allowed to cool and stopped with saturated ammoniumchloride solution. For the extraction, 250 ml of ethyl acetate wereadded and extracted 2× with ammonium chloride solution (sat.) and 1×with sodium chloride solution (sat.). The organic phase was dried withsodium sulfate and evaporated. The residue was purified by columnchromatography on silica gel 40-63 μm with the eluent ethylacetate/n-hexane+1% TEA (1:5).4,4′-di-(tert-Butoxydiphenylsilyloxy)-4″-methoxytriphenylcarbinol isobtained as a white powdery solid.

Yield: 3.00 g≈52% of theory.

R_(f) (ethyl acetate/n-hexane 1:5+1% TEA): 0.26

1.6 4,4′,4″-tri(tert-Butoxydiphenylsilyloxy)triphenylcarbinol (S6)

0.41 g (16.87 mmol, 1.2 eq) of magnesium was suspended in 20 ml of THFin a heat-dried flask under argon. 18.90 g (20.86 mmol, 1.5 eq) of4-tert-butoxydiphenylsilyloxybromobenzene were slowly added dropwisefrom a dropping funnel with continuous boiling. After the addition wascomplete, the mixture was heated with an oil bath to 85° C. and stirredfor 15 min. The resulting magnesium bromide was allowed to cool to 70°C., and 1 eq 10.10 g (13.97 mmol) of4,4′-di-tert-butoxydiphenylsilyloxybenzophenone dissolved in 50 ml ofTHF were added dropwise and heated at 70° C. for a further 2 h. Themixture was then allowed to cool and stopped with saturated ammoniumchloride solution. For the extraction, 250 ml of ethyl acetate wereadded and extracted 2× with ammonium chloride solution (sat.) and 1×with sodium chloride solution (sat.). The organic phase was dried withsodium sulfate and evaporated. The residue was purified by columnchromatography on silica gel 40-63 μm with the eluent ethylacetate/n-hexane+1% TEA (1:10).4,4′,4″-tri-(tert-Butoxydiphenylsilyloxy)triphenylcarbinol is obtainedas a white powdery solid.

Yield: 10.20 g≈68% of theory.

R_(f) (ethyl acetate/n-hexane 1:10+1% TEA): 0.23

1.7 4-(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol

6.00 ml (6.00 mmol, 1.1 eq) of 1M TBAF solution were added dropwise to2.87 g (5.41 mmol, 1 eq) of4-tert-butoxydiphenylsilyloxytriphenylcarbinol dissolved in 20 ml of THFand stirred at room temperature (RT) for 1 h. 5 ml ofpyridine/MeOH/water (3:1:1) and pyridinium-Dowex were added to stop thereaction. After 30 min, filtration and washing with pyridine werecarried out. The solution was then coevaporated with pyridine severaltimes. 1.97 g (8.12 mmol, 1.5 eq) of Nppoc-Cl dissolved in 10 ml ofmethylene chloride were added dropwise to the mixture dissolved in 60 mlof pyridine/methylene chloride (2:1) and stirred at RT for 16 h. Themixture was then concentrated and taken up in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography on silica gel 40-63 μmwith the eluent ethyl acetate/n-hexane+1% TEA (1:2).4-Nppoc-triphenylcarbinol is obtained as a solid white foam.

Yield: 2.16 g≈83% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.68

1.84-Methoxy-4′-(2-[2-nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S2)

15.70 ml (15.70 mmol, 1.1 eq) of 1M TBAF solution were added dropwise to8.00 g (14.27 mmol, 1 eq) of4-methoxy-4′-tert-butoxydiphenylsilyloxytriphenylcarbinol dissolved in50 ml of THF and stirred at RT for 1 h. The reaction was stopped byadding 20 ml of pyridine/MeOH/water (3:1:1) and pyridinium-Dowex. After30 min, filtration and washing with pyridine were carried out. Thesolution was then coevaporated with pyridine several times. 6.50 g(26.75 mmol, 1.9 eq) of Nppoc-Cl dissolved in 15 ml of methylenechloride were added dropwise to the mixture dissolved in 60 ml ofpyridine/methylene chloride (2:1) and stirred at RT for 16 h. Themixture was then concentrated and dissolved in methylene chloride, andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography by the step gradientmethod on silica gel 40-63 μm with the eluent ethyl acetate/n-hexane+1%TEA (1:3 and 1:2). 4-Methoxy-4′-Nppoc-triphenylcarbinol is obtained as asolid white foam.

Yield: 5.5 g≈75% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.54

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=17.70 (CH3-nppoc), 33.19(CH-nppoc), 55.16 (CH3-O), 72.09 (CH2-nppoc), 81.31 (C-trityl), 113.23(C-3,5-methoxyphenyl(trityl)), 120.04 (C-3,5-nppocphenyl(trityl)),124.36 (C-3-phenyl-nppoc), 126.83 (C-4-phenyl(trityl)), 127.71(C-2,6-phenyl(trityl)), 127.91 (C-6-phenyl-nppoc), 128.28(C-4-phenyl-nppoc), 129.03 (C-3,5-phenyl (trityl)), 129.11(C-2,6-nppocphenyl(trityl)), 129.35 (C-2,6-methoxyphenyl(trityl)),132.76 (C-5-phenyl-nppoc), 136.63 (C-1-phenyl-nppoc), 138.88(C-1-methoxyphenyl(trityl)), 144.87 (C-1-nppocphenyl-(trityl)), 146.75(C-1-phenyl(trityl)), 149.93 (C-2-phenyl-nppoc), 150.18 (O(CO)O), 153.35(C-4-nppoc-phenyl(trityl)), 158.71 ppm (C-4-methoxyphenyl (trityl)).

1.94,4′-Dimethoxy-4″-(2-[2-nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S3)

8.00 ml (8.00 mmol, 1.1 eq) of 1M TBAF solution were added dropwise to4.00 g (7.23 mmol, 1 eq) of4,4′-dimethoxy-4″-tert-butoxydiphenylsilyloxytriphenylcarbinol dissolvedin 20 ml of THF and stirred at RT for 1 h. The reaction was stopped byadding 10 ml of pyridine/MeOH/water (3:1:1) and pyridinium-Dowex. After30 min, filtration, washing with pyridine and concentration were carriedout. The residue was purified by column chromatography by the stepgradient method on silica gel 40-63 μm, eluent ethyl acetate/n-hexane+1%TEA (1:3 and 1:1). 0.40 g (1.70 mmol, 1.4 eq) of Nppoc-Cl dissolved in 5ml of methylene chloride was added dropwise to 0.40 g (1.25 mmol) of theresulting 4,4′-dimethoxy-4″-hydroxytriphenylcarbinol dissolved in 30 mlof pyridine/methylene chloride (2:1). The mixture was stirred at RT for16 h. It was then concentrated, dissolved in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography on silica gel 40-63 μmwith the eluent ethyl acetate/n-hexane+1% TEA (1:3).4,4′-Dimethoxy-4″-Nppoc-triphenylcarbinol is obtained as a solid whitefoam.

Yield: 0.57 g≈86% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.59

1.10 4,4′-di(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S4)

5.28 ml (5.28 mmol, 2.2 eq) of 1M TBAF solution were added dropwise to2.00 g (2.40 mmol, 1 eq) of4,4′-di-tert-butoxydiphenylsilyloxytriphenylcarbinol dissolved in 30 mlof THF and stirred at RT for 1 h. The reaction was stopped by adding 20ml of pyridine/MeOH/water (3:1:1) and pyridinium-Dowex. After 30 min,filtration and washing with pyridine were carried out. The solution wasthen coevaporated with pyridine several times. 2.19 g (9.00 mmol, 3.7eq) of Nppoc-Cl dissolved in 10 ml of methylene chloride were addeddropwise to the mixture dissolved in 60 ml of pyridine/methylenechloride (2:1) and stirred at RT for 16 h. The mixture was thenconcentrated, dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography on silica gel 40-63 μm with the eluentethyl acetate/n-hexane+1% TEA (1:1). 4,4′-di-Nppoc-triphenylcarbinol isobtained as a solid white foam.

Yield: 0.75 g≈40.9% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.55

1.114-Methoxy-4′,4″-di(2-[2-nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S5)

20.77 ml (20.77 mmol, 2.2 eq) of 1M TBAF solution were added dropwise to7.85 g (9.44 mmol, 1 eq) of4-methoxy-4′,4″-di-tert-butoxydiphenylsilyloxytriphenylcarbinoldissolved in 50 ml of THF and stirred at RT for 1 h. The reaction wasstopped by adding 20 ml of pyridine/MeOH/water (3:1:1) andpyridinium-Dowex. After 30 min, filtration and washing with pyridinewere carried out. The solution was then coevaporated with pyridineseveral times. 6.10 g (25.03 mmol, 2.6 eq) of Nppoc-Cl dissolved in 10ml of methylene chloride were added dropwise to the mixture dissolved in60 ml of pyridine/methylene chloride (2:1) and stirred at RT for 16 h.The mixture was then concentrated, dissolved in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography by the step gradientmethod on silica gel 40-63 μm with the eluent ethyl acetate/n-hexane+1%TEA (1:2, 2:3 to 1:1). 4-Methoxy-4′,4″-di-Nppoc-triphenylcarbinol isobtained as a solid white foam.

Yield: 3.0 g≈43% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.65

1.12 4,4′,4″-tri(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S6)

31.45 ml (31.45 mmol, 3.3 eq) of 1M TBAF solution were added dropwise to10.20 g (9.53 mmol, 1 eq) of4,4′,4″-tri-tert-butoxydiphenylsilyloxytriphenylcarbinol dissolved in200 ml of THF and stirred at RT for 1 h. The reaction was stopped byadding 20 ml of pyridine/MeOH/water (3:1:1) and pyridinium-Dowex. After30 min, filtration and washing with pyridine were carried out. Thesolution was then coevaporated with pyridine several times. 9.29 g(38.12 mmol, 4.0 eq) of Nppoc-Cl dissolved in 20 ml of methylenechloride were added dropwise to the mixture dissolved in 120 ml ofpyridine/methylene chloride (2:1) and stirred at RT for 16 h. Themixture was then concentrated, dissolved in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography by the step gradientmethod on silica gel 40-63 μm with the eluent ethyl acetate/n-hexane+1%TEA (1:2, to 1:1). 4,4′,4″-tri-Nppoc-triphenylcarbinol is obtained as asolid white foam.

Yield: 3.9 g≈44% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.49

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=17.68 (CH3-nppoc), 33.17(CH-nppoc), 72.14 (CH2-nppoc), 80.89 (C-trityl), 120.31(C-3,3′,3″,5,5′,5″-trityl), 124.35 (C-3-phenyl-nppoc), 127.83(C-6-phenyl-nppoc), 128.27 (C-4-phenyl-nppoc), 128.99(C-2,2′,2″,6,6′,6″-trityl), 132.77 (C-5-phenyl-nppoc), 136.62(C-1-phenyl-nppoc), 144.09 (C-1,1′,1″-trityl), 150.14 (O(CO)O), 150.17(C-2-phenyl-nppoc), 153.36 ppm (C-4,4′,4″-trityl).

MS (TOF, ES+): 952.29 [M+Na], 968.25 [M+K].

MS (TOF, ES−): 928.42 [M−H], 694.37 [M−Cl].

C₄₉H₄₃N₃O₁₆: 929.90

1.13 Halogenation and Etherification to Give5′-O-Nppoc-triphenylmethylthymidine Derivatives

1 eq of the triphenylcarbinol derivative were heated with 100 eq ofthionyl chloride at 90° C. for 3 h. Methylene chloride was then addedand heated was continued for 2 h. The mixture was then concentrated withcyclohexane 3×. The resulting triphenylmethyl chloride derivative wasdissolved in methylene chloride and, under protective gas, 2 eq ofthymidine dissolved in pyridine and methylene chloride were addeddropwise. After stirring at RT for 16 h, the reaction was stopped byadding ethanol. The mixture was then concentrated, dissolved inmethylene chloride and extracted 2× with sodium bicarbonate solution(1N) and 1× with saturated sodium chloride solution. The organic phasewas dried and evaporated. The residue was purified by columnchromatography on silica gel 40-63 μm. Mobile phases and yields are tobe found in table 1 below:

TABLE 1 Boiling time R_(f): eluent + 1% Substance SOCl₂ TEA Yield5′-O-(4-Nppoc-trityl)- 2 + 3 h (CH₂Cl₂ + 5% 95% thymidine MeOH) 0.265′-O-(4-Methoxy-4′-Nppoc- 3 + 2 h (EA/Hex 2:1) 50% trityl)thymidine 0.185′-O-(4,4′-dimethoxy- 2 + 3 h (CH₂Cl₂ + 5% 83% 4″-Nppoc-trityl)thymidineMeOH) 0.30 5′-O-(4,4′-DiNppoc-trityl) 4 + 1 h (EA/Hex 3:1) 26% thymidine0.34 5′-O-(4-methoxy- 4 + 1 h (EA/Hex 2:1) 44% 4′,4″-diNppoc-trityl)-0.21 thymidine 5′-O-(4,4′4″-triNppoc- 4 + 1 h (EA/Hex 3:1) 13%trity)lthymidine 0.25

The 5′-triphenylmethylthymidine derivatives are obtained as a solidwhite foam in yields between 13-95% of theory.

NMR Data for5′-O-(4-methoxy-4′-(2-[2-nitrophenyl)propyloxycarbonyloxy)trityl]thymidine

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=11.78 (5-CH3) 17.60 (CH3-nppoc),33.10 (CH-nppoc), 40.72 (C-2′), 55.15 (O-CH3), 63.67 (C-5′), 70.95(C-3′), 72.11 (CH2-nppoc), 84.65 (C-4′), 85.03 (C-1′), 86.64 (C-trityl),111.20 (C-5), 113.25 (C-3,5-methoxyphenyl (trityl)), 120.35(C-3,5-nppocphenyl(trityl)), 124.27 (C-3-phenyl-nppoc), 127.27(C-4-phenyl(trityl)), 127.69 (C-6-phenyl-nppoc), 127.79(C-2,6-phenyl(trityl)), 128.19 (C-4-phenyl-nppoc), 128.98(C-4-phenyl-nppoc), 129.07 (C-2,6-nppocphenyl(trityl)), 134.35(C-1-methoxyphenyl(trityl)), 135.54 (C-6), 136.52 (C-1-phenyl-nppoc),141.60 (C-1-nppocphenyl(trityl)), 144.91 (C-1-phenyl(trityl)), 149.80(C-2-phenyl-nppoc), 150.08 (O(CO)O), 150.61 (C-2), 153.31(C-4-nppocphenyl (trityl)), 158.74 (C-4-methoxyphenyl(trityl)), 164.17ppm (C-4).

1.144-Methoxy-4′-(2-[2-nitrophenyl]propyloxycarbonyloxy)triphenylcarbinol(S2)

For synthesis without protective groups, 0.50 g (20.60 mmol, 1.2 eq) ofmagnesium was suspended in 20 ml of THF in a heat-dried flask underargon. 3.2 ml (25.70 mmol, 1.5 eq) of bromoanisole were slowly addeddropwise from a dropping funnel while boiling continuously. After theaddition was complete, the mixture was heated to 85° C. with an oil bathand stirred for 15 minutes. The resulting magnesium bromide was allowedto cool to 70° C., and 3.40 g (17.17 mmol, 1 eq) of4-hydroxybenzophenone dissolved in 50 ml of THF were added dropwise andstirred at 70° C. for a further 2 h. The mixture was then allowed tocool and the reaction was stopped with saturated ammonium chloridesolution. For extraction, 250 ml of ethyl acetate were added andextracted 2× with ammonium chloride solution (sat.) and 1× with sodiumchloride solution (sat.). The organic phase was dried with sodiumsulfate and evaporated. The workup was dispensed with, and coevaporationwith pyridine was carried out several times.

11.00 g (45.00 mmol) of Nppoc-Cl dissolved in 15 ml of methylenechloride were added dropwise to the mixture dissolved in 40 ml ofpyridine/methylene chloride (1:1) and stirred at RT for 16 h. Themixture was then concentrated, dissolved in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography by the step gradientmethod on silica gel 40-63 μm, eluent ethyl acetate/n-hexane+1% TEA (1:3and 1:2). 4-Methoxy-4′-Nppoc-triphenylcarbinol is obtained as a solidwhite foam.

Yield: 3.7 g≈42% of theory.

R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.54

1.15 5′-O-(4,40,4″-tripivaloyltriphenylmethyl)thymidine from rosolicacid

10 ml (9.8 mmol, 4.7 eq) of pivaloyl acid chloride were added dropwiseto 5 g (17.22 mmol) of p-rosolic acid dissolved in 75 ml of pyridine and2.5 ml of 2,6-lutidine. The mixture was then stirred at 70° C. for 80min. On cooling, crystals formed and were isolated and washed withpyridine. 1 g (1.73 mmol) of the crystals were dissolved in 25 ml ofmethylene chloride and reacted with DMAP catalysis (20 mg, 0.17 mmol)with 20 ml of 2,6-lutidine and 220 mg (0.86 mmol) of thymidine. Themixture was then concentrated, dissolved in methylene chloride andextracted 2× with sodium bicarbonate solution (1N) and 1× with sodiumchloride solution (sat.). The organic phase was dried and evaporated.The residue was purified by column chromatography by the step gradientmethod on silica gel 40-63 μm, eluent ethyl acetate/n-hexane+1% TEA(1:3, 1:2). 5′-O-(4,4′,4″-tripivaloyltriphenylmethyl)thymidine isobtained as a solid white foam.

Yield: 0.18 g≈13.15% of theory.

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=12.11 (5-CH3) 27.02 (CH3-piv),38.93 (C-piv), 41.02 (C-2′), 63.92 (C-5′), 70.33 (C-3′), 84.53 (C-4′),85.92 (C-1′), 86.20 (C-trityl), 110.82 (C-5), 120.97(C-3,3′,3″,5,5′,5″-trityl), 129.59 (C-2,2′,2″,6,6′,6″-trityl), 135.09(C-6), 140.47 (C-1,1′,1″-trityl) 151.00 (C-2), 154.21(C-4,4′,4″-trityl), 164.10 (C-4), 176.78 ppm (C═O-piv).

1.165′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylmethyl)deoxythymidinefrom rosolic acid

10 g (34.44 mmol) of p-rosolic acid dissolved in 120 ml of pyridine and30 ml of 2,6-lutidine was heated to 50° C., and 44 g (178.28 mmol) ofNppoc-Cl were added dropwise. After reaction at 50° C. for 16 h, excesschloride was quenched by adding t-buthanol. The reaction was checked byTLC (R_(f) (ethyl acetate/n-hexane 1:1+1% TEA): 0.35). After reactionfor a further 30 min, 8.2 g of thymidine and 750 mg of DMAP were addedat a temperature of 45° C. After stirring for 16 h, the mixture wasconcentrated, dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography on silica gel 40-63 μm, eluent ethylacetate/n-hexane+1% TEA (3:1).5′-O-(4,4′,4″-tri-(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylmethyl)deoxythymidine is obtained as a solid white foam.

Yield: 10 g≈25% of theory.

R_(f) (ethyl acetate/n-hexane 3:1+1% TEA): 0.25

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=12.00 (5-CH3) 17.59 (CH3-nppoc),33.06 (CH-nppoc), 40.50 (C-2′), 63.74 (C-5′), 71.48 (C-3′), 72.14(CH2-nppoc), 84.55 (C-4′), 85.52 (C-1′), 86.16 (C-trityl), 111.13 (C-5),120.58 (C-3,3′,3″,5,5′,5″-trityl), 124.26 (C-3-phenyl-nppoc), 127.63(C-6-phenyl-nppoc), 128.19 (C-4-phenyl-nppoc), 129.58(C-2,2′,2″,6,6′,6″-trityl), 135.29 (C-6), 136.47 (C-1-phenyl-nppoc),140.53 (C-1,1′,1″-trityl), 150.09 (C-2-phenyl-nppoc), 150.34 (O(CO)O),150.34 (C-2), 153.13 (C-4,4′,4″-trityl), 163.73 ppm (C-4).

MS (TOF, ES⁺): 1 175.89 [M+Na], 1 191.83 [M+K].

MS (TOF, ES⁻): 1 152.87 [M−H].

C₅₉H₅₅N₅O₂₀: 1 154.12

1.17 5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylmethyl)-N⁴-isobutryldeoxycytidine

2.8 g (9.64 mmol) of p-rosolic acid dissolved in 20 ml of pyridine and20 ml of 2,6-lutidine were heated to 50° C., and 14.0 g (56.72 mmol) ofNppoc-Cl were added dropwise. After 30 min, excess chloride was quenchedby adding t-butanol. The reaction was checked by TLC (R_(f) (methylenechloride/methanol 5%+1% TEA): 0.36). After reaction at a temperature of45° C. for a further 30 min, 3.80 g of N⁴-isobutryldeoxycytidine and 250mg of DMAP dissolved in 30 ml of DMF were added. After 36 h, the mixturewas concentrated, dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography on silica gel 40-63 μm, eluent ethylacetate/n-hexane+1% TEA (3:1).5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)-N⁴-isobutryldeoxycytidineis obtained as a solid white foam.

Yield: 2.6 g≈16.8% of theory.

R_(f) (ethyl acetate/n-hexane 3:1+1% TEA): 0.25

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=17.61 (CH3-nppoc), 18.89 (CH3-ibu),33.10 (CH-nppoc), 36.10 (CH-ibu), 41.69 (C-2′), 63.39 (C-5′), 70.67(C-3′), 72.10 (CH2-nppoc), 85.90 (C-4′), 86.18 (C-trityl), 87.06 (C-1′),96.45 (C-5), 120.60 (C-3,3′,3″,5,5′,5″-trityl), 124.27(C-3-phenyl-nppoc), 127.62 (C-6-phenyl-nppoc), 128.21(C-4-phenyl-nppoc), 129.55 (C-2,2′,2″,6,6′,6″-trityl), 132.72(C-5-phenyl-nppoc), 136.50 (C-1-phenyl-nppoc), 140.50(C-1,1′,1″-trityl), 143.85 (C-6), 150.08 (O(CO)O), 150.08(C-2-phenyl-nppoc), 153.15 (C-4,4′,4″-trityl), 155.32 (C-2), 162.32(C-4), 176.91 ppm (C═O-ibu).

MS (TOF, ES⁺): 1 209.48 [M+H], 1 231.48 [M+Na].

MS (TOF, ES⁻): 1 209.48 [M−H].

C₆₂H₆₀N₆H₂₀: 1 209.20

1.185′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)-N⁶-pivaloyldeoxyadenosine

2.8 g (9.64 mmol) of p-rosolic acid dissolved in 20 ml of pyridine and20 ml of 2,6-lutidine were heated to 50° C., and 14.0 g (56.72 mmol) ofNppoc-Cl were added dropwise. After 30 min, excess chloride was quenchedby adding t-butanol. The reaction was checked by TLC (R_(f) (methylenechloride/methanol 5%+1% TEA): 0.36). After reaction for a further 30 minat a temperature of 45° C., 5.7 g of N⁶-pivaloyldeoxyadenosine and 250mg of DMAP dissolved in 30 ml of DMF were added. After 36 h, the mixturewas concentrated, dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography on silica gel 40-63 μm, eluent ethylacetate/n-hexane+1% TEA (3:1).5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)-N⁶-pivaloyldis obtained as a solid white foam.

Yield: 1.3 g≈6.8% of theory.

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=17.56 (CH3-nppoc) 27.40 (CH3-piv),33.04 (CH-nppoc), 39.69 (C-2′), 40.32 (C-piv), 63.73 (C-5′), 71.48(C-3′), 72.35 (CH2-nppoc) 84.45 (C-4′), 85.82 (C-trityl), 85.90 (C-1′),120.39 (C-3,3′,3″,5,5′,5″-trityl), 122.61 (C-5), 124.21(C-3-phenyl-nppoc), 127.58 (C-6-phenyl-nppoc), 128.17(C-4-phenyl-nppoc), 129.51 (C-2,2′,2″,6,6′,6″-trityl), 132.59(C-5-phenyl-nppoc), 136.67 (C-1-phenyl-nppoc), 140.68(C-1,1′,1″-trityl), 141.32 (C-8), 149.90 (C-4), 149.93(C-2-phenyl-nppoc), 150.03 (O(CO)O), 151.02 (C-6), 152.30 (C-2), 153.10(C-4,4′,4″-trityl), 175.67 ppm (C═O-piv).

MS (TOF, ES⁺): 1 247.49 [M+H], 1 270.48 [M+Na].

MS (TOF, ES⁻): 1 245.46 [M−H].

C₆₄H₆₃N₈O₁₉: 1 247.25

1.195′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)-N-2-isobutryldeoxyguanosine

4.0 g (13.8 mmol) of p-rosolic acid dissolved in 50 ml of pyridine and30 ml of 2,6-lutidine were heated to 50° C., and 18.0 g (73.9 mmol) ofNppoc-Cl were added dropwise. After 16 h, excess chloride was quenchedby adding t-butanol. The reaction was checked by TLC (R_(f) (ethylacetate/n-hexane 1:1+1% TEA): 0.36). After reaction for a further 30 minat a temperature of 40° C., 4.60 g of N²-isobutryldeoxyguanosine and 250mg of DMAP dissolved in 30 ml of DMF were added. After 36 h, the mixturewas concentrated, dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography on silica gel 40-63 μm, eluent ethylacetate/n-hexane+1% TEA (3:1).5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxycarbonyloxy)triphenylmethyl)-N²-isobutryldeoxyguanosineis obtained as a solid white foam. (Methylene chloride/methanol 5%+1%TEA): 0.22

Yield: 1.3 g≈7.0% of theory.

¹³C-NMR: (125.75 MHz, CDCl₃, TMS): δ=17.63 (CH3-nppoc) 18.83 (CH3-ibu),33.12 (CH-nppoc), 35.90 (CH-ibu), 39.89 (C-2′), 64.19 (C-5′), 70.97(C-3′), 72.10 (CH2-nppoc), 83.90 (C-4′), 85.66 (C-trityl), 85.82 (C-1′),120.31 (C-5), 120.34 (C-3,3′,3″,5,5′,5″-trityl), 124.21(C-3-phenyl-nppoc), 127.61 (C-6-phenyl-nppoc), 128.25(C-4-phenyl-nppoc), 129.57 (C-2,2′,2″,6,6′,6″-trityl), 132.78(C-5-phenyl-nppoc), 136.47 (C-1-phenyl-nppoc), 137.74 (C-8), 140.82(C-1,1′,1″-trityl), 147.91 (C-4), 148.37 (C-2), 149.92(C-2-phenyl-nppoc), 150.04 (O(CO)O), 153.19 (C-4,4′,4″-trityl), 155.94(C-6), 179.84 ppm (C═O-ibu).

C₆₃H₆₀N₈O₂₀: 1 249.22

1.205′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)-N⁶-pivaloyldeoxyadenosineβ-cyanoethyl-N,N-diisopropylaminophosphoamidite

1.0 g (0.80 mmol) of the 5′-triphenylmethyldeoxyadenosine derivativewere dissolved in methylene chloride and 2.6 eq of ethyldiisopropylamineand cooled under protective gas to 0° C. Then 1.2 eq of chlorophosphinewere added dropwise and stirred at 0° C. for 10 min. The mixture wasstirred at RT for 1 h and the reaction was stopped with ethanol. It wasthen concentrated and dissolved in methylene chloride and extracted 2×with sodium bicarbonate solution (1N) and 1× with sodium chloridesolution (sat.). The organic phase was dried and evaporated. The residuewas purified by column chromatography with methylene chloride/methanol2% on silica gel 40-63 μm.5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxycarbonyl-oxy)triphenylmethyl)-N⁶-pivaloyldeoxyadenosineβ-cyano-ethyl-N,N-diisopropylaminophosphoamidite is obtained as a solidwhite foam.

Yield: 0.7 g≈60% of theory.

³¹P-NMR (202.45 MHz, CDCl₃): δ=149.37 ppm.

1.215′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]propyloxy-carbonyloxy)triphenylmethyl)thymidineβ-cyano-ethyl-N,N-diisopropylaminophosphoamidite

2.0 g (1.73 mmol) of the 5′-triphenylmethylthymidine derivative weredissolved in methylene chloride and 2.6 eq of ethyldiisopropylamine andcooled under protective gas to 0° C. Then 1.2 eq of chlorophosphine wereadded dropwise and stirred at 0° C. for 10 min. The mixture was stirredat RT for 1 h and the reaction was stopped with ethanol. It was thenconcentrated and dissolved in methylene chloride and extracted 2× withsodium bicarbonate solution (1N) and 1× with sodium chloride solution(sat.). The organic phase was dried and evaporated. The residue waspurified by column chromatography with ethyl acetate/hexane 1:1 onsilica gel 40-63 μm.5′-O-(4,4′,4″-tri(2-[2-Nitrophenyl]-propyloxycarbonyloxy)triphenylmethyl)thymidineβ-cyano-ethyl-N,N-diisopropylaminophosphoamidite is obtained as a solidwhite foam.

Yield: 2.1 g≈89% of theory.

³¹P-NMR (202.45 MHz, CDCl₃): δ=149.77 ppm.

Example 2 Elimination of Protective Groups

The acid resistance of the nonactivated protective groups was carriedout with 80% acetic acid and 1% trichloroacetic acid (standardconditions for oligonucleotide synthesis). Table 2 shows the result ofthis study.

All the samples are initially dissolved for 300 s or 900 s in 80% aceticacid and analyzed by HPLC. Those which withstand this treatment withoutether cleavage are treated for 300 s or 900 s with 1% trichloroaceticacid and then analyzed.

TABLE 2 1% trichloroacetic Compound Symbol 80% acetic acid acid5′-O-(4-Nppoc-trityl)- S1 stable labile thymidine5′-O-(4-Methoxy-4′-Nppoc- S2 stable labile trityl)thymidine5′-O-(4,4′-Dimethoxy- S3 labile labile 4″-Nppoc-trityl)- thymidine5′-O-(4,4′-diNppoc- S4 stable labile trityl)thymidine5′-O-(4-Methoxy-4,4″- S5 stable labile diNppoc-trityl) thymidine 5′-O-S6 stable Stable (4,4′,4″-triNppoc- trityl)thymidine

It is evident that the 4,4′,4″-tri-Nppoc-trityl protective group (S6) isstable in trichloroacetic acid.

Example 3 Optimization of the Synthesis

A first optimization is achieved by using diphenylmethylchlorosilane inplace of tert-butoxydiphenylchlorosilane.

Example 4 2 Solid-Phase Experiments

The two-stage protective groups are tested on a solid phase. The test offunctionality in principle is carried out on a DNA processor D of febitag in a Geniom® one apparatus.

5′-O-(4,4′,4″-tri-Nppoc-trityl)thymidine phosphoamidite is condensedonto a silanized DNA processor surface. Spot illumination, subsequentacid treatment (max. 2 min) and coupling of a spacer are carried out. Adetection is finally carried out once again.

The analogous experiment is carried out as control. However, in place ofthe amidite with the tandem protective group, an Nppoc-dT amidite iscoupled. It was possible by this experiment to show the functionality ofthe two-stage protective group.

1. A process for the synthesis of a nucleic acid by stepwise assembly ofbuilding blocks, wherein the building blocks are building blocks for thesynthesis of a nucleic acid, wherein at least one of the building blockscarries a two-stage protective group, wherein the two-stage protectivegroup contains a photoactivatable group selected from the groupconsisting of nitroveratryloxycarbonyl (NVOC),α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 3,5-dimethoxybenzoincarbonate (DMBOC), 2-(o-nitrophenyl)propyloxycarbonyl(NPPOC), o-nitrobenzyl and 2-(o-nitrophenyl) ethyl, wherein thephotoactivable group is removed by an illumination step and theremainder of the two-stage protective group is removed by a subsequentacid treatment step.
 2. The process as claimed in claim 1, characterizedin that the building block with the two-stage protective group has thegeneral formula I:

where R₁ and R₂ are each independently selected from hydrogen, OR_(3,)O(CH₂) _(n)COOR₃ and NHZ, R₃ comprises a C₁-C₈ alkyl group, a C₂-C₈alkenyl group, a C₂-C₈ alkynyl group or/and C₆-C₂₀ aryl group which mayoptionally have substituents, X is the building block, Y is in each caseindependently the photoactivatable group, Z is an amino-protectivegroup, n is an integer from 0 to 4, and where R₁ or/and R₂ mayoptionally be replaced by Y.
 3. The process as claimed in claim 2,characterized in that a two-stage protective group which carries atleast one fluorescent group is used.
 4. The process as claimed in claim3, characterized in that Y, R₃ or/and Z carry the fluorescent group. 5.The process as claimed in claim 1, characterized in that the buildingblocks are phosphoramidites.
 6. The process as claimed in claim 5,characterized in that phosphoramidite building blocks carry thetwo-stage protective group on the 5′-O atom.
 7. The process as claimedin claim 1, wherein at least one of the building blocks contains aspacer or linker group.
 8. The process as claimed in claim 1,characterized in that the synthesis is carried out on a solid phase. 9.The process as claimed in claim 8, characterized in that asite-dependent synthesis of a plurality of nucleic acids, each with adifferent nucleotide sequence, is carried out on a single support.
 10. Acompound of the general formula I:

where R₁ and R₂ are each independently selected from hydrogen, OR_(3,)O(CH₂) _(n)COOR₃ and NHZ, R₃ comprises a C₁-C₈ alkyl group, a C₂-C₈alkenyl group, a C₂-C₈ alkynyl group or/and C₆-C₂₀ aryl group which mayoptionally have substituents, X is a building block for the synthesis ofa nucleic acid or a leaving group, Y is in each case independently aphotoactivatable group, Z is an amino-protective group, n is an integerfrom 0 to 4, where R₁ or/and R₂ may optionally be replaced by Y, andwherein the photoactivatable group is selected from the group consistingof nitroveratryloxycarbonyl (NVOC), α-methyl-6-nitropiperonyloxycarbonyl(MeNPOC), 3,5-dimethoxybenzoincarbonate (DMBOC),2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl and2-(o-nitrophenyl) ethyl and wherein the photoactivatable group isremovable by illumination.
 11. The compound as claimed in claim 10,characterized in that it carries at least one fluorescent group.
 12. Thecompound as claimed in claim 11, characterized in that Y, R₃ or/and Zcarry a fluorescent group.
 13. A method of synthesizing a nucleic acidfrom building blocks, wherein at least one of the building blocks isprotected by a two-stage protective group, wherein the at least one ofthe building blocks protected by a two-stage protective group is acompound of Formula (I), said method comprising the steps of: removing aphotoactivatable group of the two-stage protective group byillumination, and subsequently removing the rest of the two-stageprotective group from the building block by acid treatment whereinformula (I) is

where R₁ and R₂ are each independently selected from hydrogen, OR_(3,)O(CH₂) _(n)COOR₃ and NHZ, R₃ comprises a C₁-C₈ alkyl group, a C₂-C₈alkenyl group, a C₂-C₈ alkynyl group or/and C₆-C₂₀ aryl group which mayoptionally have substituents, X is a building block for the synthesis ofa nucleic acid or a leaving group, Y is in each case independently aphotoactivatable group, Z is an amino-protective group, n is an integerfrom 0 to 4, where R₁ or/and R₂ may optionally be replaced by Y, andwherein the photoactivatable group is selected from the group consistingof nitroveratryloxycarbonyl (NVOC), α-methyl-6-nitropiperonyloxycarbonyl(MeNPOC), 3,5-dimethoxybenzoincarbonate (DMBOC),2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl and2-(o-nitrophenyl) ethyl and wherein the photoactivatable group isremovable by illumination.
 14. The process of claim 2, wherein thesubstituent is selected from the group consisting of a halogen, —OH,—SH, —O—, —S(O)—, —S(O)₂—, —NO_(2,) —CN and —NHZ, wherein Z is anamino-protective group.
 15. The compound of claim 10, wherein thesubstituent is selected from the group consisting of a halogen, —OH,—SH, —O—, —S(O)—, —S(O)₂—, NO_(2,) —CN and —NHZ, wherein Z is anamino-protective group.
 16. The method of claim 13, wherein thesubstituent is selected from the group consisting of a halogen, —OH,—SH, —O—, —S(O)—, —S(O)₂—, —NO_(2,) —CN and —NHZ, wherein Z is anamino-protective group.
 17. The method of claim 13, characterized inthat the compound of formula I carries at least one fluorescent group.18. The method of claim 17, characterized in that Y, R₃ or/and Z carry afluorescent group.
 19. The compound of claim 10, characterized in thatthe building block is a phosphoramidite.
 20. The compound of claim 19,characterized in that the linkage with the phosphoramidite buildingblock is through the 5′-O atom.
 21. The process of claim 13,characterized in that the building blocks are phosphoramidites.
 22. Theprocess of claim 21, characterized in that phosphoramidite buildingblocks carry the two-stage protective group on the 5′-O atom.